The objective of this research proposal is to identify and characterize functional sites in E. coli rRNA which are involved in the process of protein synthesis. Using a plasmid-borne rrnB operon we are producing mutations in the 16S and 23S rRNA genes by in vitro and in vivo methods. These mutations will be studied by a variety of molecular and biochemical techniques. Sites of interest include the Shine-Dalgarno region near the 3' 16S rRNA that base-pairs to mRNA and may bs involved in frameshifting, a potential site in 16S rRNA for the recognition of a translational enhancer in mRNA, regions in 16S and 23S rRNA involved in subunit association, and sites in 16S rRNA potentially involved in switching between the active and inactive forms of the 30S subunit. Additional regions to be studied include putative tRNA binding and decoding sites as well as potential sites for missense suppression, regions in 16S and 23S rRNA which confer resistance to antibiotics such as spectinomycin, streptomycin, the amino-glycosides, and thiostrepton, and finally regions in 16S rRNA which are involved in termination of translation. Lethal mutants will bs produced on plasmids containing repressible promoters PL and T7. The plasmid- coded rRNAs will be characterized by maxicells as well as by a variety of functional assays which measure rate and fidelity of translation in vivo, including nonsense and missense suppression. Methods will be used to specifically isolate the plasmid-coded mutant ribosomes for structural and functional studies using silent mutations in 16S (at 1192) and 23S (at 1067) rRNA that confer resistance to spectinomycin and thiostrepton, respectively. These studies should contribute to our understanding of the functional role of rRNA during protein synthesis by defining the catalytic activity of particular regions of the RNA at a molecular level.